Link to the University of Pittsburgh Homepage
Link to the University Library System Homepage Link to the Contact Us Form

Single Molecule Studies of DNA Damage Recognition by XPA

Beckwitt, Emily (2019) Single Molecule Studies of DNA Damage Recognition by XPA. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

Download (27MB) | Preview


Nucleotide excision repair (NER) is responsible for the repair of a wide range of DNA lesions, including UV-induced photoproducts and bulky base adducts. XPA is an essential protein in eukaryotic NER, although questions about its stoichiometry and mechanism of damage recognition have been heretofore unresolved. Regions of intrinsic disorder within the N- and C-termini of XPA have made structural work on the full-length protein challenging and compel an alternative approach. We have used PeakForce Tapping® atomic force microscopy to show that human XPA binds to DNA as a monomer and bends it ~60°. Furthermore, XPA demonstrated specificity for the helix-distorting base adduct, N-(2’-deoxyguanosin-8-yl)-2-acetylaminofluorene. Single molecule fluorescence microscopy revealed that DNA-bound XPA exhibits multiple modes of linear diffusion between paused phases. These included long distance motion with rapid diffusion (D ≈ 0.04 μm2/s) consistent with hopping and short distance motion (D ≈ 0.0003 μm2/s) consistent with sliding along the DNA contour. The presence of DNA damage increases pausing by proteins undergoing one-dimensional target search. A truncated mutant, lacking most of the intrinsically disordered regions and made up of just residues 98-239 of the DNA binding domain, exhibits less pausing on UV-damaged DNA compared to the full length protein. In summary, our data are consistent with a model in which the conformational state of XPA is dependent upon the presence of DNA damage and bending.


Social Networking:
Share |


Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Beckwitt, Emilybeckwitt@pitt.edubeckwitt0000-0001-7052-5820
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Thesis AdvisorVan Houten,
Committee ChairOpresko,
Committee MemberBruchez,
Committee MemberWang,
Committee MemberBrodsky,
Date: 16 December 2019
Date Type: Publication
Defense Date: 8 November 2019
Approval Date: 16 December 2019
Submission Date: 5 December 2019
Access Restriction: 1 year -- Restrict access to University of Pittsburgh for a period of 1 year.
Number of Pages: 196
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Molecular Biophysics and Structural Biology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: XPA, Single molecule, Atomic force microscopy, Fluorescence microscopy, Protein-DNA interaction, Nucleotide excision repair
Date Deposited: 17 Dec 2019 04:41
Last Modified: 16 Dec 2020 06:15


Monthly Views for the past 3 years

Plum Analytics

Actions (login required)

View Item View Item